The broad, long-term objective of this proposal is to understand the mechanism by which precursors of messenger RNAs (pre-mRNAs) are spliced. The focus of this application is on a biochemical approach to identify novel interactions and factors. This will be addressed in mono-intronic pre-mRNAs and in pre-mRNAs that have a choice between alternative sets of splice site partners. The specific aims of this proposal are: Specific aim no. 1. Characterization of pre-commitment and commitment complexes. We propose to determine the molecular basis for the interaction between the U1 snRNP particle and ASF/SF2, specifically the role of the 70 kDa protein of U1. We propose to investigate the relationship of these U1/ASF complexes to the functional commitment complex. We will also study the interactions of U2AF and other factors known to recognize cis-acting elements at or near the 3' splice site with the U1/ASF complexes. We propose to identify and purify new factors required for commitment. Specific aim no. 2. Characterization of metazoan splicing commitment complexes involved in alternative splicing. The Sxl regulated system will be utilized to study the formation of alternative commitment complexes. Specific aim no. 3. Characterization and purification to homogeneity of a novel mammalian factor that is required for formation of the spliceosome from the pre-spliceosome. Specific aim no. 4. Characterization and purification to homogeneity of a factor that is required for the second step of splicing.